Occurrence ofPenicillium brocaeand，andPenicillium citreonigrum单击功能区上，which Produce a Mutagenic Metabolite and a Mycotoxin Citreoviridin，Respectively，in Selected Commercially Available Rice Grains in Thailand

Abstract

公共的，可操作的，可操作的，可操作的Penicilliumspecies responsible for toxic yellowed rice。Penicilliumspecies were obtained from seven out of 10rice samples tested.Among them，onePenicillium citreonigrumisolate and sixPenicillium brocaeisolates were morphologically identified.TheP.citreonigrumisolate produced the mycotoxin citreoviridin on ayeast extract sucrose broth medium.Mycotoxin surveys showed that citreoviridin was not detected in any samples，but one out of 10rice samples ted was positive foraflatoxin B_1个at a level of 5.9μg/kg.An Ames test revealed that methanol extracts from rice grains inoculated with selectedP.brocaestrains TA 100 and YG7108 of isolates were positive for strains TA 100 and YG7108 ofSalmonella型phimurium单击功能区上，suggesting the presence of base-pair substitution and DNA alkylation mutagens.Our data obtained here demonstrated that aflatoxin B_1个and toxicP.citreonigrumwere present on domestic rice grains in Thailand，although limited samples were tested。Penicillium brocae单击功能区上，which may produce mutagenic metabolites，was isolated for the first time from the surface of Thai rice grains。

1.Introduction

Fungal contamination in food and foodstuffs causes a number of problems in commercially available products，such as food spoilage and food poisoning.In terms of fungi，mycotoxin contamination is of great concern to human health around the world.In Thailand，the major mycotoxins associated with cancer risk found in market commodities are aflatoxin（AF）B_1个and other aflatoxins[1个]。AFB_1个为应对国际Agency for Research on Cancer，and several studies have reported rice contaminated with AFB_1个and other aflatoxins among Thai commodities[2单击功能区上，3单击功能区上，4个]。in the postharvest condition，harvested rice grins may be damaged in several ways，such as fungal invasion，mycotoxin contamination，physicochemical changes，and insect activity.Particularly，fungal invasion of stored agricultural products leads to a loss of quality in the storage environment5]。However，there is currently insufficient information on stored rice as an agricultural commodity in Thailand。

A staple cereal plant，rice（Oryza sativaL.）is widely cultivated in Asia，North and South America，the European Union，and Middle Eastern and African countries.Rice provides20%of the world’s dietary energy suply，while wheat and maize supply19%and5%，respectively[6个]。The nutritive contents of rice vary depending on the type of rice，but rice is known to be a good source of thiamine，riboflavin and niacin，along with some kinds of amino acids.Thus，the contamination of rice grains with mycotoxins is a serious problem for human health[7]。内钢筋混凝土，the contamination of rice withAspergillusand，andFusariumtoxins such as aflatoxins，trichothecenes，and fumonisins，has been of great concern[8个单击功能区上，9]，but little attention has been paid toPenicilliumtoxin due to its low presence in rice。

Before and after World War II，the toxic rice problems in Japan occurred after rice was imported，and three historical cases have been documented[10单击功能区上，11]。安装was called“yellow rice”and was caused by contamination of rice grains with toxigenicPenicilliumspecies.The first case was the isolation ofPenicillium citreonigrum（formerly，P.citreo-viride）。Fungus-damaged rice was imported from Taiwan in 1937，and found to be contaminated with the toxic metabolite citreoviridin（CTV），which has been suggested to be responsible for beriberi disease.The second case was an occurrence ofPenicillium islandicum（current taxon，Talaromyces islandicus）in a batch of not yellowed but yellowish-brown rice。This rice was imported from Egypt in 1948.Mycotoxin studies have revealed thatP.islandicumproduces hepatotoxic mycotoxins，such as luteoskyrin，and cyclochlorotine.The third case was caused by the citrininin-producerPenicillium citrinum.The rice was imported from Thailand in 1951[12]。Citrinin is nephrotoxic，but has low acute toxicity.These toxicological characteristics，including the induction of cancer and chemical structures，have been reported by Japan ese mycotoxin researchers[10单击功能区上，12]。

Recently，anoutbreak of beriberi disease occurred in the state of Maranhao，Brazil，and1207cases were reported，including40deaths.In rice samples from the outbreak region，the neurotoxic mycotoxin CTV was detected at ranges from 0.9 to 31.1μg/kg.CTV is a known toxic metabolite ofP.citreonigrum车辆安全系统，车辆安全系统13]。However，the relationship between CTV and the disease is still unclear。

In the present study，we examined rice commodities in Thai markets for surveillance of mycoflora.In addition to the alcohol treatment，the rice grains were ted without any treatment in order to assess the stored rice environment in Thailand.Particularly，the presence of the toxic yellowed fungusP.citreonigrumand related species were surveyed.These monoverticillate species are characterized by slow growth and yellow pigment production on the surface or reverse of the colonies on Czapek yeast agar（CYA）medium。The isolates obtained were tested for CTV production.As an indicator of human toxicity，their metabolites were assayed for mutagenic activities using an Ames test.Contamination of Thai rice samples with mycotoxins was also analyzed。

2.Results

2.1。ycoflora on Rice Grains

Based on macroscopic observation，the developed fungi were classified simply as genera of fungi.In the alcohol-treated group，the developed fungi wereAspergillus单击功能区上，Eurotium（teleomorph of xerophilicAspergillus），ucorand，andRhizopusshown as Zygomycetes in火焰1单击功能区上，and other fungi.Similar findings were noted in the untreated group，except forPenicilliumalso being observed.As shown in火焰1单击功能区上，the frequency of the genera differed depending on the rice type and region of Thailand.However，almost the same major fungal genera developed between the groups，except for the genusPenicillium.Isolates ofPenicilliumspecies were only found on untreated rice grains.In addition，in the sample from the Surin region，monoverticillate and yellow pigment-producingPenicilliumisolates were observed.These isolates were subjected to identification at the species level。

2.2。Morphological and Phylogenetic Identification

动力输送和动力输送Penicilliumisolates were cultured on CYA and malt extract agar（MEA）media at25°C for seven days。基本功系数Penicilliumisolates，one isolate was identified asP.citreonigrum单击功能区上，双稳态功率Penicilliumisolates could not be determined by morphological observation alone.Therefore，these isolates were subjected to a molecular phylogeny analysis。

火焰2shows the NJ-tree inferred from theβ-tubulin gene（β-tub）的边缘Penicilliumspecies.Each reference species formed a monophyletic clade with high bootstrap support.TheP.citreonigrumisolate identified morphologically belonged toPenicillium toxicarium单击功能区上，which is included in the clade ofP.citreonigrum.Recently，P.toxicariumwas moved toPenicillium citreosulfuratumby molecular phylogeny alone[14]，并进行isolate was suggested to identify asP.citreosulfuratum.However，due to finely roughened conidia，especially with roughness arranged in undulation，the isolate differed from the IMI92228 strain，which is an ex-type ofP.citreosulfuratum单击功能区上14单击功能区上，15]。Thus，the isolate was identified asP.citreonigrumbased on the criterion ofPenicilliumtaxonomy by Pitt[15]。Two unknownPenicillium基础施工方案Penicillium brocae基础上的phylogenetic analysis（火焰2）。热morphological characteristics of these isolates were then compared with the description ofP.brocaereported by Peterson et al.[16]。

As shown inFigure3单击功能区上，P.brocaeisolates grew slowly when cultured on CYA at25°C for seven days。Colonies were23-25mm in diameter，grayish blue-green，velvety，and centrally produced moderate to abundant yellow exudate.The reverse colorwas yellowish brown or brownish purple，producing soluble naphthalene yellow pigment at the edge.The isolates did not grow at37°C。Conidiophores were short and smooth-walled，and vesiculate，monoverticillate penicillus was observed.Phialides were ampulliform，8-9×2-3μm。Conidia were spherical，2.5-3.5µm in diameter，with slightly roughened walls，forming short chains.These characteristics of the observedP.brocaeisolates were almost the same as the original description of the species。

The sequence data ofP.citreonigrumAFS-0075，andP.brocaeAFS-0006and AFS-0073 strains were deposited in the DNA Data Bank of Japan（DDBJ）under the accession numbers LC216411，LC216406，and LC216408，respectively。

As relatively fewPenicilliumspecies were observed，an additional trial was conducted in the same sample but using different rice grains.The trial showed that noP.citreonigrumisolate and fourP.brocaeisolates were obtained.Among them，threeP.brocaeisolates were subjected to the molecular phylogeny analysis.The identification of the isolates was confirmed using theβ-tubregion（火焰2）。The sequence data ofP.brocaeAFS-0009，AFS-0089，and AFS-0090 strains were deposited in the DDBJ under the accession numbers LC216407，LC216409，and LC216410，respectively.The strains obtained were not used for further experiments。

2.3。utagenicity of P.brocae Metabolites

The AFS-0006and AFS-0073 strains ofP.brocaewere cultured on 20g of rice grains at25°C for14 days。Early in the culture，the rice was stained yellow，and then covered with abundant green conidia.The cultures were extracted with100mL of methanol by sonication for20min.The methanol extracts were evaporated to dryness under a nitrogen gas flow and resolved with5mL of dimethylsulfoxide。

The assay dose was calculated from the extract amounts per4g of rice grains and prepared with serial dilutions.Assays were performed with and without S9 mix using the tester strains ofSalmonella型phimuriumand，andEscherichia coli.Results showed that the methanol extracts of both strains were positive against the TA 100 and YG7108 strains ofS、S。typhimurium单击功能区上，as base pair substitution mutations occurred primarily at one of the G-C pairs.The mutagenic activity of YG7108was higher than that of TA 100。火焰4shows the dose-response curve of mutagenicity of the methanol extracts from theP.brocaeAFS-0006and AFS-0073 strains forS.typhimuriumYG7108，both with and without rat liver S9mix.The mutagenicity increased dose dependently，especially when treated with the S9mix.These data indicated the presence of alkylating agents in theP.brocaemetabolites。

2.4。ycotoxin Analysis

For theP.citreonigrumisolate obtained，an LC-QTOF analysis revealed that the isolate produced high amounts of CTV，when cultured on ayeast extract sucrose（是）liquid cultural medium。火焰5and，and火焰6show the total ion chromatogram of the fungal extract，the accurate mass spectra of the main peak of the fungal extract，and citreoviridin A standard solution，respectively.The relative mass error of the protonated molecular ion at main peak was2.71ppm。

Multiple mycotoxins survey showed that CTV was negative for any samples of Thai rice grains，although CTV-producing fungus was found.Among the 10rice samples tested，AFB_1个was detected at a concentration of 5.9µg/kg in sample No.8obtained from the Pathum Thani region。火焰7AFB为shows extracted ion chromatograms of the rice samples for_1个detection.The other mycotoxins examined were not detected in any samples（data not shown）。

3.Discussion

不均匀进场应力测量occurred in 1937 in Japan and was found to have been caused byP.citreonigrum单击功能区上12]。The fungus produced atoxic metabolite，CTV，which is responsible for acute cardiac beriberi[第十七节：]。As beriberi disease did not occur in Japan after World War II，this yellowed rice toxic syndrome was largely forgotten.However，in2006to2008，anoutbreak occurred in Brazil due to the consumption of CTV-contaminated rice[18]。This incident reminded us of past issues associated with yellowed rice in Japan。

In this study，we obtained the target fungusP.citreonigrumfrom the surface of rice grains in Thai commodities.In addition，we also isolatedP.brocaefrom the same samples，which was unexpected，asP.brocaeis regarded as an endophytic fungus.These twoPenicilliumspecies showed slow growth on culture media。

In general，fungal isolation in rice grains is conducted with surface disinfection using chlorine，as several kinds of air-borne fungi may be present on the surface of rice grains.These contaminants can interfere with the surveillance of mycoflora on rice grains.However，we compared two methhods the prent， surface disinfection and direct plating without disinfection in order to examine the surface mycobiota on rice grains.We found toxigenicP.citreonigrumand endophyticP.brocae，along with otherPenicilliumspecies，only in the untreated group（火焰1）。As these twoPenicilliumspecies are not common air-borne fungi，our untreated approach seems to have been useful for the isolation of uncommonPenicilliumspecies，possibly due to their susceptibility to the disinfecting agents compared with the common field or storage fungi ofAspergillus单击功能区上，Bipolaris单击功能区上，Colletotrichum单击功能区上，Curvularia单击功能区上，ucor单击功能区上，Phoma，andRhizopus单击功能区上19]。This may explain why previous studies on mycoflora in rice failed to find anyP.citreonigrum或，或P.brocae单击功能区上，except in the case of theP.citreonigrum可恢复的，可恢复的18]。

An isolate ofP.citreonigrumidentified morphologically was obtained from arice sample in the Surin region，which is in the northeast of Thailand，close to Cambodia.It produced CTV on YES medium.Kim et al.[20已报告的thatP.toxicariumwas an endophytic fungus，because it was isolated from healthy needles ofPinus rigida，along withPenicillium fellutanum。P.citreonigrumand，andP.toxicarium预应力混凝土火焰2），but the taxon remains unclear。P.brocaewas isolated firstly from the coffee berry borerHypothenemus hampei单击功能区上16]，but it known to be an endophytic fungus，generally derived from the coffee plant[21]，or theZyzzyasp.sponge[22]，或the marine mangrove plantAvicennia marina单击功能区上23]。Two research reports have shown that insects carried experimentally toxigenicAspergillusand，andPenicillium存储maize kernels[第二十四节：“并加热线”25]。Therefore，althoughP.citreonigrumand，andP.brocaewere both isolated from rice grains in this study，they may have been introduced to the rice by insects，such as grain weevils，which tend to favor rice.Further research will be needed to confirm our speculation。

In the mutagenicity test，twoP.brocaestrains were tested to determine whether or not they could produce mutagens.The methanol extracts obtained from moldy rice grains were positive for base-pair substitution mutation and DNA alkylation usingS.typhimuriumstrains TA100and YG7108，respectively.Interestingly，these strains suggested the mutation of GC sites.As the mutagenic activity in the YG7108strain was high，the data revealed that the metabolites ofP.brocaeAFS-0006and AFS-0073 strains induced the alkylation of the GC sites（火焰4）。Further studies are underway to clarify the mutagenic agents produced byP.brocae。

全封闭式防波堤Penicilliumspecies were present in commercially available Thai rice grains.However，CTV produced byP.citreonigrumwas negative in any rice samples tested.AFB_1个alone was detected in the No.8rice sample，although toxigenicAspergillusspecies were not found in the sample（火焰1）。The culture plate of the No.8 sample was fully covered withucor或，或Rhizopus.Anti-fungal chemicals，dichloran and rose bengal were inactive in depressing the growth of these fungi in the sample.Aflatoxin contamination in Thai commodities has been reported by several studies[2单击功能区上，8个]。In this study，wealso detected AFB_1个in a sample of Luem phua rice in the Pathum Thani region，close to Bangkok.Although our survey was admittedly limited，the level of AFB_1个contamination was higher than that in previous reports.Levels of contamination with AFB_1个may depend on rice types。

4.Conclusions

Commercially available Thai rice samples were contaminated with several fungal species.Most of the species observed were not toxic，but a few toxigenic fungi were present，such asP.citreonigrumand，andP.brocae.Of note，these toxigenicPenicilliumspecies possessed good ability to produce CTV or a mutagen.Thus，these species could be a potential threat to human health if environmental conditions are suitable for their growth。

5.Materials and Methods

5.1。样品和Fungal Strain

Ten samples of rice were purchaed from nine markets in Thailand.The types of rice and market regions were as follows:Jasmine rice（No.1）in Bangkok，Dong Ruk rice（No.2）in Surin，Pratum rice（No.3）in Ayuddhaya，Riceberry gaba（No.4）in Nakorn Pathom，Jasminrice and No（No.5）.10）in Chiang Rai，Red jasmine rice（No.6）in Chaiyaphum，Organic brown rice（No.7）in Chaing Mai，Luem phua rice（No.8）in Pathum Thani，and Riceberry（No.9）in Petchabune。These rice samples were chosen randomly，and packed into sealed plastic bags.The bags were kept closed until use.The samples sizes of the rice grains were ca.1kg each。

As reference strain，theP.citreo-virideIMI92228 strain was obtained from the culture collection of CABI in the United Kingdom。

5.2。Fungal Isolation and Morphological Identification

One hundred rice grains from all10 samples were used，and treated in two different manners:50 grains were exposed to 70%ethanol solution for 30s and then washed twice with sterile distilled water，and the other50 grains were not treated in any manner.Following this treatment，five rice grains per plate were placed onto dichloran rose bengal chloramphenicol agar（DRBC）or dichloran18%glycerol agar（DG18）medium and cultured at25°C for5days。After incubation，the genera of the developed fungi were simply classified bya macroscopic observation[26]。Among them，Penicilliumspecies，showing monoverticillate and yellow pigmentation，were transferred to CYA and MEA media（BD，Sparks，MD，USA）and cultured at25°C for7days。According to the taxonomic criterion of thePenicilliumspecies[26]，the isolates were morphologically identified based on colony textures and microscopic observation。

5.3。Molecular Phylogenetic Analysis

amolecular phylogenetic analysis was carried out as follows.Fungi were pre-cultured on potato dextrose（PD）broth medium（Difco Laboratories，Franklin Lakes，NJ，USA）at25°C for3-4 days。The genomic DNA was extracted from fungal mycelia by the sodium dodecyl sulfate method[27]。Theβ-tubregions were chosen for the phylogenic analysis.PCR amplification was done using Bt2a and Bt2b primers[28]。Sequencing was performed using a BigDy Terminator v3.1 Cycle Sequencing Kit and ABI3130 DNA Analyzer（Applied Biosystems，Foster City，CA，USA）according to the manufacturer’s instructions.Forty-five sequences ofβ-tub雷吉斯of the variousPenicilliumspecies andAspergillus nigerwere downloaded from GenBank and used as reference data for the phylogenetic analysis.The species were selected with regard to variousPenicilliumclades reported by Visagie et al.[29]andA.nigerwas used as an outer species.The sequences were automatically aligned and the alignments were manually adjusted using MEGA6[30]。Aphylogenetic tree was constructed with the neighbor joining method by the maximum composite likelihood model using MEGA6。

5.4。utagenicity Assay

热mutagenicity assay was performed usingS.typhimuriumstrains TA98、TA100、TA1537、and YG7108、andE.colistrain WP2/uvrA/pKM101with and without rat liver S9mix.These strains were chosen for detecting frameshift mutations（TA98 and TA1537）and base-pair substitution mutations（TA100，YG7108and WP2），and YG7108was particularly sensitive for DNA alkylation.The assay was tested for methanol extracts obtained from moldy rice grains，which were individually inoculated with tester isolates and cultured at25°C for14 days。accordance with the official guide of the mutagenicity test using microorganisms[31]，which was revised by the Ministry of Health，Labor and Welfare of Japan in 1991，along with the method of Yamada et al.[32为了…，为了S、S。typhimuriumYG7108strain，the assay was performed at five doses with two plates per dose for each sample.In addition，mutagenic standard chemicals were used as positive references against tester strains.The experiments were repeated twice， and the mutagenic activity was calculated from the slope of the linear portion of the dose-response curve using the statistical model of least squares linear regression。

5.5。ycotoxin Analysis

For the CTV analysis，the tester isolate was cultured on YES liquid medium at25°C for 10days。The CTV extract was then obtained in accordance with the method described by da Rocha et al.[13]。In target MS/MS acquisition for CTV，a data rate of three mass spectra per sec in MS/MS mode was used.Target compounds were detected and reported from accurate-mass data using Agilent Mass Hunter Qualitive and The Agilent Mycotoxins and Related Metabolited Personal Compound Databary and Databary（PCDL）software proms。The PCDL database contains455target compound names，moleculae，CAS numbers，structure and 297MSspectra.The Qualitative Analysis software program tentatively identified target compounds using a“Find by Formula”algorithm with the following criteria:relative ss marror，5ppm；isotope abundance and spacing score，greater than90out of 100.The reagent of citreoviridin A standard used was obtained from Fermentek Ltd.（Jerusalem，Israel）。

Fifty grams of rice grains from each sample were powdered using a blender machine，and a7.5g portion of the powder sample was weighed and transferred to a50-mL centrifuge tube.Mycotoxins were extracted using the QuEChERS extraction kits（Agilent Technologies，Inc.，Tokyo，Japan）with slightly modified citrate buffer.For clean-up，adispersive solid-phase extraction（Agilent Technologies，Inc.）was used.In accordance with the manufacturer’s protocol，the purified extracts obtained were analyzed by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry（LC-QTOFMS）equipped with a dual-spray Jet Stream electrospray ionization source。代理技术1290 Infinity II LC系统，Inc.）consisting of binary pump，degasser，column compartment and autosampler with a reverse-phase column of ZORBAX Eclipse plus C₁₈（100×2.1mm，1.8μm）and operated with a gradient mobile phase containing 0.1%formic acid+10mM ammonium acetate（A）and acetonitrile，or methanol（B）。The initial combination was90%A and 10%B，changing linearly to 0%A and 100%B over30min.The flow rate was0.2mL/min。

Agilent Technologies，Inc.）operated in positive ion mode with a mass ranging from100to1000Da and a data rate of 1.5 mass spectra per sec.The instrument acquired data using the following parameters:drying gas temperature，200℃；drying gas flow，20L/min；nebulizer，50psi；sheath gas temperature，400℃；sheath gas flow，12L/min；VCap.4000V；nozzle，0V；fragmentor，360V；skimmer，65V；and octapole RF peak，750.A constant flow of Agilent TOF reference solution containing purine（m/z=121.05087）和HP-921（m/z=922.00979）through the reference nebulizer allowed the system to continuously correct for any mass drift。

Twenty-six mycotoxins；AFB_1个，AFB₂，AFG_1个，AFG₂，AFM_1个，citrinin，CTV，cyclochlorotine，cyclopiazonic acid，deoxynivalenol（DON），DON-glucoside，3-acetylDON，15-acetylDON，diacetoxyscirpenol，fumonisin B_1个，fumonisin B₂，luteoskyrin，nivalenol（NIV），NIV-glucoside，4-acetylNIV，ochratoxin，patulin，sterigmatocystin，T-2toxin，HT-2toxin，and zearalenone，were examined by LC-QTOFMS。